Sleep disordered breathing induced by cervical spinal cord injury and effect of adenosine A1 receptors modulation in rats.

Sleep-disordered breathing (SDB) is very common after spinal cord injury (SCI). The present study was designed to evaluate the therapeutic efficacy of adenosine A1 receptor blockade (8-cyclopentyl-1,3-dipropylxanthine, DPCPX) on SDB in a rodent model of SCI. We hypothesized that SCI induced via left hemisection of the second cervical segment (C2Hx) results in SDB. We further hypothesized that blockade of adenosine A1 receptors following C2Hx would reduce the severity of SDB. In the first experiment, adult male rats underwent left C2Hx or sham (laminectomy) surgery. Unrestrained whole body plethysmography (WBP) and implanted wireless electroencephalogram (EEG) were used for assessment of breathing during spontaneous sleep and for the scoring of respiratory events at the acute (~1 wk), and chronic (~6 wk) time points following C2Hx. During the second experiment, the effect of oral administration of adenosine A1 receptor antagonist (DPCPX, 3 times a day for 4 days) on SCI induced SDB was assessed. C2Hx animals exhibited a higher apnea-hypopnea index (AHI) compared with the sham group, respectively (35.5 ± 12.6 vs. 19.1 ± 2.1 events/h, P < 0.001). AHI was elevated 6 wk following C2Hx (week 6, 32.0 ± 5.0 vs. week 1, 42.6 ± 11.8 events/h, respectively, P = 0.12). In contrast to placebo, oral administration of DPCPX significantly decreased AHI 4 days after the treatment (159.8 ± 26.7 vs. 69.5 ± 8.9%, P < 0.05). Cervical SCI is associated with the development of SDB in spontaneously breathing rats. Adenosine A1 blockade can serve as a therapeutic target for SDB induced by SCI.NEW & NOTEWORTHY The two key novel findings of our study included that 1) induced cervical spinal cord injury results in sleep-disordered breathing in adult rats, and 2) oral therapy with an adenosine A1 receptor blockade using DPCPX is sufficient to significantly reduce apnea-hypopnea index following induced cervical spinal cord injury.

Colocalization of A2a but not A1 adenosine receptors with GABA-ergic neurons in cardiopulmonary chemoreflex network in the caudal nucleus of the solitary tract.

Adenosine operating in the nucleus of the solitary tract (NTS) may inhibit or facilitate neurotransmitter release from nerve terminals and directly inhibit or facilitate central neurons via A1 and A2a pre- and postsynaptic receptors, respectively. However, adenosine A2a receptors, may also activate GABA-ergic neurons/terminals which in turn inhibit glutamatergic transmission in the NTS network. Our previous studies showed that adenosine operating via both A1 (inhibitor) and A2a (activator) receptors powerfully inhibits the cardiopulmonary chemoreflex (CCR) at the level of the caudal NTS. A1 receptors most likely inhibit glutamate release in the CCR network, whereas A2a receptors facilitate NTS GABA-ergic mechanisms which in turn inhibit CCR glutamatergic transmission. Therefore, we hypothesized that A2a receptors are located on NTS GABA-ergic neurons/terminals whereas A1 receptors may be located on NTS glutamatergic neurons/terminals. We investigated this hypothesis using double immunofluorescent staining for A2a or A1 adenosine receptors and GABA synthesizing enzyme, GAD67, in 30 µm thick, floating, medullary rat sections. We found that A2a adenosine receptors are localized within the GABA-ergic cells in the caudal NTS, whereas A1 adenosine receptors are absent from these neurons. Instead, A1 receptors were located on non-GABA-ergic (likely glutamatergic) neurons/terminals in the caudal NTS. These data support our functional findings and the hypothesis that adenosine A2a, but not A1 receptors are located on GABA-ergic neurons.

Transporter Protein-Coupled DPCPX Nanoconjugates Induce Diaphragmatic Recovery after SCI by Blocking Adenosine A1 Receptors.

Respiratory complications in patients with spinal cord injury (SCI) are common and have a negative impact on the quality of patients' lives. Systemic administration of drugs that improve respiratory function often cause deleterious side effects. The present study examines the applicability of a novel nanotechnology-based drug delivery system, which induces recovery of diaphragm function after SCI in the adult rat model. We developed a protein-coupled nanoconjugate to selectively deliver by transsynaptic transport small therapeutic amounts of an A1 adenosine receptor antagonist to the respiratory centers. A single administration of the nanoconjugate restored 75% of the respiratory drive at 0.1% of the systemic therapeutic drug dose. The reduction of the systemic dose may obviate the side effects. The recovery lasted for 4 weeks (the longest period studied). These findings have translational implications for patients with respiratory dysfunction after SCI. SIGNIFICANCE STATEMENT: The leading causes of death in humans following SCI are respiratory complications secondary to paralysis of respiratory muscles. Systemic administration of methylxantines improves respiratory function but also leads to the development of deleterious side effects due to actions of the drug on nonrespiratory sites. The importance of the present study lies in the novel drug delivery approach that uses nanotechnology to selectively deliver recovery-inducing drugs to the respiratory centers exclusively. This strategy allows for a reduction in the therapeutic drug dose, which may reduce harmful side effects and markedly improve the quality of life for SCI patients.

Systemic administration of rolipram increases medullary and spinal cAMP and activates a latent respiratory motor pathway after high cervical spinal cord injury.

BACKGROUND/OBJECTIVE: High cervical spinal cord hemisection interrupts descending respiratory drive from the rostral ventral respiratory group in the medulla to the ipsilateral phrenic motoneurons. Hemisection results in the paralysis of the ipsilateral hemidiaphragm. Chronic administration of rolipram, a specific phosphodiesterase-IV inhibitor, promotes synaptic plasticity and restores phrenic nerve function after a high cervical spinal cord lesion. Here, we test the hypothesis that an acute administration of rolipram will increase spinal and medullary levels of 3',5'-cyclic adenosine monophosphate (cAMP) and induce phrenic nerve recovery after cervical (C2) spinal cord hemisection. METHODS: Male Sprague-Dawley rats were subjected to left C2 hemisection surgery 1 week before experimentation. Bilateral phrenic nerve activity was recorded in anesthetized, vagotomized, and pancuronium paralyzed rats, and rolipram was intravenously applied (2 mg/kg). RESULTS: Intravenous administration of rolipram increased phrenic nerve output in uninjured control and left C2 spinal cord-hemisected rats. In addition, rolipram restored respiratory-related activity to the left phrenic nerve made quiescent by the hemisection. In both uninjured and hemisected rats, rolipram significantly enhanced phrenic inspiratory burst amplitude and burst area compared with predrug values. Also, rolipram concomitantly increased spinal and medullary cAMP. CONCLUSIONS: These results suggest that a phosphodiesterase inhibitor capable of elevating cAMP levels can enhance phrenic nerve output and restore respiratory-related phrenic nerve function after high cervical spinal cord injury. Thus, targeting the cAMP signaling cascade can be a useful therapeutic approach in promoting synaptic efficacy and respiratory recovery after cervical spinal cord injury.

The crossed phrenic phenomenon and recovery of function following spinal cord injury.

This review will focus on neural plasticity and recovery of respiratory function after spinal cord injury and feature the "crossed phrenic phenomenon" (CPP) as a model for demonstrating such plasticity and recovery. A very brief summary of the earlier literature on the CPP will be followed by a more detailed review of the more recent studies. Two aspects of plasticity associated with the CPP that have been introduced in the literature recently have been spontaneous recovery of ipsilateral hemidiaphragmatic function following chronic spinal cord injury and drug-induced persistent recovery of the ipsilateral hemidiaphragm lasting long after animals have been weaned from drug treatment. The underlying mechanisms for this plasticity and resultant recovery will be discussed in this review. Moreover, two new models involving the CPP have been introduced: a mouse model which now provides for an opportunity to study CPP plasticity at a molecular level using a genetic approach and light-stimulated induction of the CPP accomplished by transfecting mammalian cells with channelrhodopsin. Both models provide an opportunity to sort out the intracellular signaling cascades that may be involved in motor recovery in the respiratory system after spinal cord injury. Finally, the review will examine developmental plasticity of the CPP and discuss how the expression of the CPP changes in neonatal rats as they mature to adults. Understanding the underlying mechanisms behind the spontaneous expression of the crossed phrenic pathway either in the developing animal or after chronic spinal cord injury in the adult animal may provide clues to initiating respiratory recovery sooner to alleviate human suffering and eventually eliminate the leading cause of death in human cases of spinal cord injury.

Spinal cord injury in neonates alters respiratory motor output via supraspinal mechanisms.

Upper cervical spinal cord injury (SCI) alters respiratory output and results in a blunted respiratory response to pH/CO2. Many SCI studies have concentrated on respiratory changes in neural function caudal to injury; however few have examined whether neural plasticity occurs rostral to SCI. Golder et al. (2001a) showed that supraspinal changes occur to alter respiratory output after SCI. Furthermore, Brown et al. (2004) showed that neural receptors change rostral to a thoracic SCI. We hypothesized that SCI in neonates will alter supraspinal output, show a blunted response to pH and alter receptor protein levels in the medulla. On postnatal day 0/1, a C2 SCI surgery was performed. Two days later, neonates were anesthetized and brainstem-spinal cords removed. Respiratory-related activity was recorded using the in vitro brainstem-spinal cord preparation and the superfusate pH was changed (pH 7.2, 7.4 and 7.8). The respiratory-like frequency was significantly reduced in SCI rats indicating supraspinal plasticity. Increasing the pH decreased respiratory-like frequency and peak amplitude in injured and sham controls. Increasing the pH increased burst duration and area in sham controls, whereas in injured rats, the burst duration and area decreased. Western blot analysis demonstrated significant changes in glutamate receptor subunits (NR1, NR2B and GluR2), adenosine receptors (A1, A2A), glutamic acid decarboxylase (65) and neurokinin-1 receptors in medullary tissue ipsilateral and contralateral to injury. These data show that supraspinal plasticity in the respiratory system occurs after SCI in neonate rats. The mechanisms remain unknown, but may involve alterations in receptor proteins involved in neurotransmission.

Adenosinergic mechanisms underlying recovery of diaphragm motor function following upper cervical spinal cord injury: potential therapeutic implications.

OBJECTIVES: In adult rats, a latent respiratory motor pathway can be pharmacologically activated with 1,3-dimethylxanthine (theophylline) to restore respiratory-related activity to a hemidiaphragm paralysed by an ipsilateral upper cervical (C2) spinal cord hemisection. The purpose of this review is to describe mechanisms that underlie theophylline-induced recovery of respiratory-related function following C2 hemisection and to underscore the therapeutic potential of theophylline therapy in spinal cord injured patients with respiratory deficits. METHODS: Theophylline mediates recovery of respiratory-related activity via antagonism of central adenosine A(1) receptors. When administered chronically, the drug restores and maintains recovered function. Since theophylline is an adenosine receptor antagonist with affinity for both the adenosine A(1) and A(2) receptors, we assessed the relative contributions of each receptor to functional recovery. While A(1) receptor antagonism plays a predominant role, activation of the A(2) receptors by specific agonists subserves the A(1) receptor-mediated actions. That is, when an adenosine A(2) receptor agonist is administered first, it primes the system such that subsequent administration of the A(1) antagonist induces a greater degree of recovered respiratory activity than when the antagonist alone is administered. RESULTS: Chronic oral administration of theophylline in C2 hemisected animals demonstrates that even when animals have been weaned from the drug, theophylline-induced recovered respiratory actions persist. This suggests that in clinical application, it may not be necessary to maintain patients on long-term theophylline. We have shown that recovery of respiratory-related activity in the ipsilateral phrenic nerve can occur spontaneously 3-4 months after C2 hemisection. Theophylline administration after this post-injury period obliterates/negates the recovery function. This indicates strongly that there is therapeutic window (more acutely after injury) for the initiation of theophylline therapy. We have also demonstrated that peripheral (carotid bodies) adenosine A(1) receptors can be selectively activated to modulate theophylline-induced CNS actions. Blocking central adenosine receptors while simultaneously activating peripheral adenosine receptors minimizes the potential of respiratory muscle fatigue with theophylline. DISCUSSION: The significance of the current findings lies in the potential clinical application of theophylline therapy in spinal cord injured patients with respiratory deficits. The ultimate goal of theophylline therapy is to wean ventilator-dependent patients off ventilatory support. Thus far, our animal studies suggest that the onset of theophylline therapy must be soon after injury.

Recovery of respiratory function following C2 hemi and carotid body denervation in adult rats: influence of peripheral adenosine receptors.

The efficacy of the methylxanthine, theophylline, as a respiratory stimulant has been demonstrated previously in an animal model of spinal cord injury. In this model, an upper cervical (C2) spinal cord hemi paralyzes the ipsilateral hemidiaphragm. Theophylline restores respiratory-related activity in the paralyzed hemidiaphragm via activation of a latent respiratory motor pathway. Antagonism of central adenosine A1 receptors mediates this action. Theophylline also enhances respiratory frequency, f, defined as breaths per minute. Thus, long-term use may result in respiratory muscle or motoneuron fatigue particularly after spinal cord injury. We assessed the effects of an adenosine A1 receptor agonist, N6-p-sulfophenyladenosine (p-SPA) on theophylline's action in our model under standardized recording conditions. Four groups of rats, classified as hemisected/nonhemisected with the carotid bodies denervated (H-CBD or NH-CBD), and hemisected/nonhemisected with the carotid bodies intact (H-CBI or NH-CBI ) were used in the study. Eight days after recovery from carotid denervation, a left C2 hemi was performed in H-CBD rats. C2 hemi was also performed in H-CBI animals, and 24 h later, electrophysiologic experiments on respiratory activity were conducted in both groups of animals. Two groups using nonhemisected controls were also employed as described above. In H-CBD rats, theophylline significantly (P < 0.05) enhanced f and induced respiratory-related activity in the previously quiescent left phrenic nerve. In NH-CBD rats, theophylline significantly enhanced f. In both H-CBD and NH-CBD rats, p-SPA (0.25 mg/kg) did not significantly change theophylline-induced effects. In H-CBI rats, theophylline significantly (P < 0.05) enhanced f and induced activity in the previously quiescent left phrenic nerve. In H-CBI rats, p-SPA reduced the values to pre-theophylline discharge levels. Recovered activity was not obliterated with the agonist. In NH-CBI rats, p-SPA reduced theophylline-induced effects to pre-drug discharge levels. Adenosine A1 and A2A receptor immunoreactivity was detected in the carotid bodies. The significance of our findings is that theophylline-induced effects can be normalized to pre-drug levels by the selective activation of peripheral adenosine A1 receptors. The therapeutic benefits of theophylline, i.e., recovered respiratory function after paralysis, however, persists. The potential therapeutic impact is that respiratory muscle fatigue associated with long-term theophylline use may be minimized by a novel therapeutic approach.

Effects of long-term theophylline exposure on recovery of respiratory function and expression of adenosine A1 mRNA in cervical spinal cord hemisected adult rats.

Our lab has previously shown that when administered acutely, the methylxanthine theophylline can activate a latent respiratory motor pathway to restore function to the hemidiaphragm paralyzed by an ipsilateral C2 spinal cord hemisection. The recovery is mediated by the antagonism of CNS adenosine A1 receptors. The objective of the present study was to assess quantitatively recovery after chronic theophylline administration, the effects of weaning from the drug, and the effects of the drug on adenosine A1 receptor mRNA expression in adult rats subjected to a C2 hemisection. Rats subjected to a left C2 hemisection received theophylline orally for 3, 7, 12, or 30 days and were classified as 3D, 7D, 12D, or 30D respectively. Separate groups of 3D animals were weaned from drug administration for 7, 12, and 30 days before assessment of respiratory recovery. Additional groups of 7D and 12D animals were also weaned from drug administration for 7 and 12 days prior to assessment. Sham-operated controls received theophylline vehicle for similar periods. Quantitative assessment of recovered respiratory activity was conducted under standardized electrophysiologic recording conditions approximately 18 h after each drug application period. Serum theophylline analysis was conducted at the end of electrophysiologic recordings. Adenosine A1 receptor mRNA expression in the phrenic nucleus was assessed with in situ hybridization and immunohistochemistry. Chronic theophylline induced a dose-dependent effect on respiratory recovery over a serum theophylline range of 1.2-1.9 microg/ml. Recovery was characterized as respiratory-related activity in the left phrenic nerve and expressed as a percentage of activity in the homolateral nerve in noninjured animals under similar recording conditions. Recovered activity was 34.13 +/- 2.07, 55.89 +/- 2.96, 74.78 +/- 1.93, and 79.12 +/- 1.75% respectively in the 3D, 7D, 12D, and 30D groups. Theophylline-induced recovered activity persisted for as long as 30 days when drug administration was stopped and serum levels of the drug were virtually undetected. Furthermore, recovered activity in 3D and 7D animals increased significantly as a function of duration of weaning. Adenosine A1 receptor mRNA expression was not significantly changed by theophylline administration. It is concluded that recovery of respiratory function in C2-hemisected rats induced by chronic theophylline is unrelated to adenosine A1 receptor mRNA expression. Recovered activity persists even when drug administration has been stopped. The significance of our results is that in the clinical application of theophylline to improve respiratory impairment, intermittent drug administration may be sufficient to engender and maintain the therapeutic benefits of the drug.

Adenosine A1 receptor mRNA expression and the effects of systemic theophylline administration on respiratory function 4 months after C2 hemisection.

BACKGROUND: Previous studies from our laboratory have demonstrated that in an animal model of acute cervical spinal cord injury (SCI), respiratory function can be restored by theophylline. We also have shown that respiratory recovery occurs spontaneously after prolonged postinjury survival periods when a hemidiaphragm is paralyzed by an ipsilateral upper cervical (C2) spinal cord hemisection. Theophylline mediates functional recovery by central nervous system adenosine A1 receptor antagonism; however, it is unclear whether adenosine receptors are altered after prolonged postinjury periods and whether theophylline can further enhance restored respiratory function that occurs spontaneously. OBJECTIVE: To assess putative effects of systemic theophylline administration on further enhancing spontaneous respiratory muscle recovery 4 months after C2 hemisection in rats and to determine whether adenosine A1 receptor mRNA expression is altered in these animals. METHODS: Electrophysiologic assessment of respiratory activity in the phrenic nerves was conducted in C2 hemisected rats 4 months after hemisection under standardized conditions. Immediately thereafter, rats were killed and the cervical spinal cords were prepared for adenosine A1 receptor mRNA expression by in situ hybridization. RESULTS: Spontaneous recovery of respiratory activity in the ipsilateral phrenic nerve was detected in a majority (15/20) of C2 hemisected animals and amounted to 44.06% +/- 2.38% when expressed as a percentage of activity in the homolateral phrenic nerve in noninjured animals. At the optimal dosage used in the acute studies, theophylline (15 mg/kg) did not enhance, but rather unexpectedly blocked, recovered respiratory activity in 4 out of 5 animals tested. At dosages of 5 mg/kg and 2.5 mg/kg, the drug blocked recovered respiratory activity in 3 out of 4 and 3 out of 5 animals tested, respectively. Quantitative analysis of adenosine A1 receptor mRNA expression did not reveal a significant difference between experimental animals and sham-operated animals. CONCLUSION: The blockade or attenuation of spontaneously recovered respiratory activity following theophylline administration cannot be attributed to changes in adenosine A1 receptors because there were no significant differences in adenosine A1 mRNA expression with sham-operated animals. Lack of alteration in A1 mRNA expression 4 months after cervical SCI suggests that A1 receptor plasticity is not activated by chronic injury. Obliteration of spontaneous recovery with theophylline most likely involves a separate unknown mechanism. These findings suggest that there may be a limited therapeutic window for the clinical application of theophylline in SCI patients with respiratory deficits. Theophylline may be more effective clinically in the acute phase of injury rather than in the chronic phase.

Theophylline-induced respiratory recovery following cervical spinal cord hemisection is augmented by serotonin 2 receptor stimulation.

Cervical spinal cord hemisection leads to a disruption of bulbospinal innervation of phrenic motoneurons resulting in paralysis of the ipsilateral hemidiaphragm. We have previously demonstrated separate therapeutic roles for theophylline, and more recently serotonin (5-HT) as modulators to phrenic nerve motor recovery; mechanisms that likely occur via adenosine A1 and 5-HT2 receptors, respectively. The present study was designed to specifically determine if concurrent stimulation of 5-HT2 receptors may enhance motor recovery induced by theophylline alone. Adult female rats (250-350 g; n=7 per group) received a left cervical (C2) hemisection that resulted in paralysis of the ipsilateral hemidiaphragm. Twenty-four hours later rats were given systemic theophylline (15 mg/kg, i.v.), resulting in burst recovery in the ipsilateral phrenic nerve. Theophylline-induced recovery was enhanced with the 5-HT2A/2C receptor agonist, (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI; 1.0 mg/kg). DOI-evoked augmentation of theophylline-induced recovery was attenuated following subsequent injection of the 5-HT2 receptor antagonist, ketanserin (2.0 mg/kg). In a separate group, rats were pretreated with ketanserin, which did not prevent subsequent theophylline-induced respiratory recovery. However, pretreatment with ketanserin did prevent DOI-induced augmentation of the theophylline-evoked phrenic nerve burst recovery. Lastly, using immunocytochemistry and in situ hybridization, we showed for the first time a positive co-localization of adenosine A1 receptor mRNA and immunoreactivity with phrenic motoneurons of the cervical ventral horns. Taken together, the results of the present study suggest that theophylline may induce motor recovery likely at adenosine A1 receptors located at the level of the spinal cord, and the concurrent stimulation of converging 5-HT2 receptors may augment the response.